1. Field of the Invention
The present invention relates generally to reagents for determining kinase and phosphatase activity, and more specifically to chimeric proteins containing two fluorescent proteins and a phosphorylatable domain, and methods of using such chimeric proteins to detect kinase or phosphatase activity.
2. Background Information
Phosphorylation is the most important way that individual proteins are post-translationally modified to modulate their function, while practically all signal transduction involves dynamics of protein-protein interaction. Phosphorylation is catalyzed and controlled by kinases such as Calcium modulated kinase (CaM kinase, such as CaMKII), protein kinase A (PKA), protein kinase C (PKC), and other kinases. Various technologies have been used to enumerate the main phosphorylation/dephosphorylation events and interacting protein partners involved in cell function, including, for example, function of cardiomyocytes and B lymphocytes. However, the most common currently used technologies such as two dimensional gel electrophoresis, mass spectrometry, co-immunoprecipitation assays, and two-hybrid screens require destroying large numbers of the cells or transferring genes to heterologous organisms. As such, these methods have poor temporal and spatial resolution, and are insufficient to directly probe physiological functions such as contracture or chemotaxis, which occur on the time scale of milliseconds to minutes.
The most widely used method for detecting phosphorylation of specific proteins in single cells utilizes antibodies that discriminate between the phosphorylated and dephosphorylated forms of an antigen. Such antibodies can, in principle, reveal the phosphorylation state of the endogenous protein just prior to the time the cells were fixed for examination, without any introduction of exogenous substrates. However, the identification of antibodies that can discriminate between a phosphorylated and unphosphorylated form of a protein is time consuming and expensive. In addition, the necessary immunocytochemistry is tedious, and is difficult to reassemble into a quantitative time course.
PKC is known to play a key role in maintaining balance between normal growth and transformation (Nishizuka, 1995). PKC function in cells is exquisitely controlled by three major mechanisms: phosphorylation, required for catalytic competence, membrane-targeting, required for conformational activation, and protein:protein interactions which poise the enzyme at specific intracellular locations (Mellor and Parker, 1998; Newton, 2002b). Pertubation of any of these mechanisms disrupts cell function by altering the degree of substrate phosphorylation. A fluorescent protein, Green Fluorescent Protein (GFP) has been used to visualize translocation of PKC to membranes upon generation of diacylglycerol (DAG) and increases in calcium in living cells. (Oancea and Meyer, 1998; Sakai et al., 1997; Shirai et al., 1998b). These studies have revealed a wealth of information on the kinetics and localization of PKC inside the cell. However, GFP-labeling has not proven sufficient to determine the activation state of PKC or PKC-substrate phosphorylation. However, translocation and activation are different processes. Thus, it is not clear to what extent visualization of PKC translocation provides a measure of PKC activation or of PKC substrate phosphorylation.
Previous techniques for imaging protein heterodimerization in single cells have included observing luminescence resonance energy transfer (LRET) from a lanthanide donor attached to an antibody against one member of the heterodimer to a red dye attached to an antibody against the other partner (Root, Proc. Natl. Acad. Sci., USA 94:5685-5690, 1997). This approach has the same advantages and disadvantages as phosphorylation-specific antibodies, including it is applicable to examining endogenous proteins in intact nontransfected tissues, but has poor time resolution and difficulty in generating a continuous time course. Another mode of energy transfer is bioluminescence resonance energy transfer, in which the donor is a luciferase and the acceptor is a GFP (Xu et al., Proc. Natl. Acad. Sci., USA 96:151-156, 1999). However, although emission from multiple cells may be detectable, the feebleness of bioluminescence would be expected to make the technique difficult or impossible to use with single mammalian cells, especially if high spatial resolution is desired.
Additionally, reporters have been designed that alter fluorescence resonance energy transfer (FRET) between fluorescent proteins (Miyawaki and Tsien, 2000) or the intrinsic fluorescent properties of a fluorescent protein (Llopis et al., 1998; Nagai et al., 2001). Reporters based on FRET between fluorescent proteins can be used to glean information from living cells, provided that such reporters do not significantly perturb cell function (for example by buffering of cell signals resulting from reporter overexpression), and provided reporter specificity is maintained in cells. FRET reporters for kinase activity have been described (Sato et al., 2002; Ting et al., 2001; Zhang et al., 2001).
In order to achieve dynamic recording of phosphorylation in single cells, peptides have been labeled with acrylodan, a probe whose fluorescence can be sensitive to the phosphorylation of the peptide. For example, when acrylodan was attached to a peptide from myosin light chain, an approximately 40% decrease in emission peak amplitude upon phosphorylation in vitro was observed. When microinjected into fibroblasts, the peptide incorporated into stress fibers, but no dynamic changes were observable. Substrates for CaMKII and PKA also have been labeled with acrylodan and, after exposure to the kinase, fluorescence was about 200% and 97%, respectively, of initial values. These peptides were hydrophobic enough to stain live cells, and local intensity changes of up to 10% to 20% of initial fluorescence were seen in some regions. The fluorescence of the PKA substrate simultaneously decreased in the cytosol and increased in the nucleus by an amount that was greater than could be explained by the in vitro sensitivity, indicating that more complex factors such as translocation were dominating.
Although the use of acrylodan-labeled peptides provides no rational mechanism for phosphorylation sensitivity, the approach of developing phosphorylation-sensitive fluorescent substrates may be worth pursuing. Thus, a need exists for phosphorylation-sensitive indicators that can be used to detect phosphorylation or dephosphorylation events in a cell. The present invention satisfies this need and provides additional advantages.